DESCRIPTION: The long term goal of this application is to define the molecular mechanism for RNA recognition in RNA binding domains (RBDs). There are over 150 known examples of RBDs in proteins that regulate RNA events as diverse as pre-mRNA splice site selection, translation initiation, cap binding, mRNA transport, and mRNA stability. Because these homologous proteins are active in so many fundamental RNA-based processes, it is important to understand how they discriminate among RNA substrates. The human U1A protein is associated with the U1 snRNP (small nuclear RiboNucleoprotein Particle). The interaction between the N-terminal RBD1 of U1A with its U1 snRNA hairpin has become a paradigm for these RNA:protein complexes. However, the description of the molecular basis of RNA recognition is far from complete. The association includes conformational changes of both RNA and protein to form sequence-specific contacts, with correspondingly complex energetics, as well as many undefined physicochemical interactions. These experiments are designed to define the mechanism of U1A RBD1:RNA recognition, with the expectation that other RBDs will show similar features. Pairwise coupling theory will be used to identify the origins of the local cooperativity that creates the protein binding surface. Specific electrostatic contributions to the association are assessed through incorporation of methyl phosphonates and mutation of specific charged amino acids. Binding must require intrinsic conformational flexibility of both RBD1 and RNA; their local flexibility is modulated by mutation and measured by 15N/13C NMR relaxation. Structural perturbations to RBD1 that alter its stability will be described using NMR in conjunction with computational homology modeling to correlate global RBD structure with RNA binding. Finally, a model system will be developed to test the functional requirements of protein structure and sequence, consisting of a minimal RBD1 composed of a three-stranded anti-parallel beta-sheet with C- and N-terminal tails; the structure of a stable complex with RNA will be solved by NMR.